KIF2C accelerates the development of non-small cell lung cancer and is suppressed by miR-186-3p via the AKT-GSK3β-β-catenin pathway

This study aimed to explore how kinesin family member 2C (KIF2C) influences the progression of non-small cell lung cancer (NSCLC). The levels of KIF2C and microRNA-186-3p (miR-186-3p) were examined by quantitative real-time polymerase chain reaction (qRT-PCR). Through the utilization of cell counting kit-8 (CCK-8) assay, colony formation assay, wound closure assay, and Transwell assay, NSCLC cell proliferation, migration, and invasion were identified, respectively. NSCLC cell apoptosis was assessed using the TUNEL assay and flow cytometry (FCM) assay. Luciferase reporter analysis was used to investigate the relationship between KIF2C and miR-186-3p. Western blot assays were conducted to investigate the influence of KIF2C on the AKT-GSK3β-β-catenin pathway. The results showed that KIF2C was up‐regulated in NSCLC cells, which predicted poor prognosis. KIF2C overexpression promoted the proliferation, migration, and invasion of NSCLC cells as well as inhibited NSCLC cell apoptosis. KIF2C was as a key target of miR-186-3p. High expression of KIF2C, meanwhile, increased the levels of β-catenin, p-GSK-3β and phosphorylated protein kinase B (p-AKT). KIF2C downregulation and miR-186-3p upregulation reversed these outcomes. As an oncogenic factor, KIF2C is negatively regulated by miR-186-3p and participates in the progression of NSCLC through the AKT-GSK3β-β-catenin pathway.

www.nature.com/scientificreports/ The regulatory involvement of miR-186-3p in NSCLC, however, remains unknown. The aim of this study was to clarify the ability of KIF2C to enhance cancer cell viability, proliferation and invasion in NSCLC by activating the AKT-GSK3β-β-catenin pathway. At the same time, miR-186-3p can negatively regulate the expression of KIF2C, thereby exerting a tumor suppressor effect 10 . Our research revealed the mechanism of KIF2C on NSCLC and the regulatory effect of miR-186-3p on KIF2C, which provides a novel therapeutic target for NSCLC.

Materials and methods
Cell culture and transfection. Human immortalized normal lung epithelial cell line BEAS-2B and lung cancer cell lines, including A549, A549-DDP, LLC, PC-9, SHP-77, NCI-H1703 and human embryonic kidney cell line 293T were purchased from the Type Culture Collection of the Chinese Academy of Sciences (Shanghai, China). Cisplatin (DDP) was first synthesized by M. Peyrone in 1845, and later proved to have a killing effect on NSCLC cells. It is now recommended for adjuvant chemotherapy in NSCLC patients after tumor resection. A549 cells are a human adenocarcinoma cell line, while A549-DDP refers to a human adenocarcinoma cell line resistant to DDP. A549-DDP cells were cultured in RPMI-1640 medium (Gibco, BRL, USA). Others were maintained in DMEM medium (Gibco, BRL, USA) at 37 °C in the humidified air with 5% CO 2 .
Apoptosis assay. Apoptosis in cells was measured using the Annexin-V-FITC/PI apoptosis detection kit (BD Biosciences, San Jose, CA, USA) in accordance with the manufacturer's instructions. Then the cell suspension was shaken thoroughly for 15 min at room temperature in darkness and supplemented with 10 μL propidium iodide (Sigma Aldrich, St. Louis, USA). Flow cytometry (FCM) was then used to analyze apoptotic cells. Three different runs of each experiment were conducted. A TUNEL assay kit (cat. no. MK1015; Wuhan Boster Biological Technology, Ltd.) was used to detect apoptotic cells according to the manufacturer's instructions. Cells were incubated overnight at 4 °C with anti-neuronal nuclei antibody (cat. no. BM4354; 1:100; Wuhan Boster Biological Technology, Ltd.) according to the manufacturer's instructions. The cells were incubated with TUNEL reaction mixture for 1 h at 37 °C before being washed three times with PBS. Images were captured using an inverted fluorescence microscope at high magnification (400×). Images in three randomly selected areas were quantitated, and the positive cells were measured using Image J software.
Transwell assay. Transwell migration chambers (8-μm pore size; Millipore, Billerica, MA, USA) were used to measure cell migration. A total of 2 × 10 5 cells were suspended in the serum-free DMEM media and planted into the top chamber together. In the lower chamber, DMEM medium with 10% FBS was added. The chambers were incubated for 48 h before removal, and crystal violet was used to stain these cells from the lower chamber following 4% paraformaldehyde for 15 min. Random six fields at high magnification (Olympus, Tokyo, Japan) were chosen, and cell migration was calculated. Additionally, the invasion assays were carried out in Transwell chambers covered with matrigel (Clontech, CA, USA). www.nature.com/scientificreports/ Colony formation assay. At a cell density of 1000 cells/dish, the transfected cells were seeded in triplicate into 60 mm cell culture plates. After 14 days of growth in DMEM medium containing 10% FBS, cells were fixed and stained with 0.1% crystal violet for 30 min. After that, the colonies were meticulously cleaned with PBS until the background was completely clear. The colony formation rate was calculated as follows: Wound healing assay. Transfected cells were planted in six-well plates for 24 h to form a monolayer. After that, each well was scratched with a cross using a sterile pipette tip held vertically. Subsequently, cells were washed with phosphate-buffered saline (PBS) and shaken for 5 min to remove the detached cells. Then the diluted samples and fresh medium were incubated for 72 h. After washing cells with PBS, add the preheated medium or a fresh sample, and take images. A Keyence BZ-9000 microscope (Keyence, Neu-Isenburg, Germany) was used to observe and capture the scratch closure at intervals of 24 h at low magnification.
Western blot. After cells were collected and fixed, the protein was extracted and examined according to the manufacturer's instructions. Put simply, the lysates were prepared with RIPA lysis buffer (Beyotime Biotechnology, Beijing, China) and centrifuged for 10 min at 4 °C at 12,000 rpm, and the BCA protein assay kit (Pierce, WI, USA) was applied to determine protein concentration in the supernatants. The sample mixed with loading buffer was added to 10% SDS-PAGE and then transferred to a nitrocellulose membrane. The primary antibodies in this part include the Anti-KIF2C antibody, Anti-p-AKT antibody, Anti-AKT antibody, Anti-p-GSK-3β antibody, Anti-GSK-3β antibody, Anti-β-actinin antibody, and Anti-GAPDH antibody. The above antibodies were purchased separately from Cell Signaling Technology (CST, MA, USA). The membranes were washed after being incubated with the primary antibodies overnight and treated for an hour with a secondary antibody labeled with horseradish peroxidase (HRP). The signal was detected with chemiluminescence phototope-HRP kit (Pierce, Rockford, USA USA) after washing the membrane.

Statistical analysis.
The results were presented as means and standard deviation. Statistics were analyzed using SPSS 20.0 (Chicago, USA). Differences between the groups were compared using the student t-test.

Results
NSCLC with a higher expression of KIF2C has a poor outcome. The purpose of this study was to determine KIF2C expression levels in human lung epithelial cells BEAS-2B and different types of NSCLC cell lines, such as mouse lung adenocarcinoma LLC, human lung adenocarcinoma cells A549, PC-9, SHP-77, A549-DDP, NCI-H1703, through qRT-PCR. In comparison with BEAS-2B, KIF2C expression was significantly higher in NSCLC cells (Fig. 1a). At the same time, KIF2C mRNA levels have been analyzed in human lung adenocarcinomas (LUAD), lung squamous cell carcinomas(LUSC), and normal lung tissue in the Cancer Genome Atlas (TCGA) dataset. According to the results, tumor tissues had significantly higher levels of KIF2C mRNA than normal tissues (Fig. 1b,c). Both high KIF2C expression in LUAD and LUSC tissues were associated with worse prognosis according to the TCGA database (Fig. 1d,e). KIF2C affects biologically malignant behaviors of NSCLC cellsWith the popularization of computed tomography and other imaging screening methods, adenocarcinoma incidence has increased significantly and has surpassed squamous carcinoma as the most common type. Adenocarcinoma is an invasive carcinoma, which is more likely to relapse and metastasize than squamous carcinoma and difficult to treat 11 . Therefore, we selected LUAD in NSCLC as the research object. Results have showed that A549-DDP cells showed the highest KIF2C expression among NSCLC cells, whereas its corresponding A549 cells showed a relatively low KIF2C expression. As a result, we used plasmid to overexpress KIF2C in low-expressing A549 cells and used small interfering RNA (siRNA) to knock down KIF2C in high-expressing A549-DDP cells. The transfection efficiencies of KIF2C overexpression and silencing were determined by qRT-PCR (Fig. 2a,b). In addition, CCK8 and flow cytometry (FCM) assays were performed to assess the proliferative ability of NSCLC cells transfected with KIF2C and KIF2C siRNA. The results showed that overexpression of KIF2C significantly promoted proliferation and inhibited apoptosis, while silencing of KIF2C inhibited proliferation and promoted apoptosis (Fig. 2c-f). Cancer cell proliferation, migration and invasion are a significant part of cancer progression, so we investigated the effect of KIF2C on these features in NSCLC by performing transwell, colony formation and wound healing assays. We performed cell proliferation, migration, and invasion assays to investigate the biological function of KIF2C in controlling the biologically malignant behaviors of NSCLC cells. The results revealed that, in comparison to the control group, KIF2C overexpression significantly promoted cell proliferation, migration, and invasion, whereas KIF2C knockdown significantly inhibited above cell behavior (Fig. 2g-i). (c.d) CCK8 and Colony formation assays were performed to assess the proliferative ability transfected with KIF2C and KIF2C siRNA plasmids; (e,f) Flowcytometry assays was used to detect cell apoptosis of overexpression and silence of KIF2C; (g,h,i) The effects of KIF2C on migration and invasion were evaluated by the wound healing and transwell assays (*P < 0.05, **P < 0.01, and ***P < 0.001).  12,13 .Therefore, we investigated if KIF2C could impact the AKT-GSK3β-β-catenin pathway via Western blotting assays. The results demonstrated that overexpression of KIF2C increased β-catenin, p-GSK-3β and p-AKT levels, while the result of KIF2C knockdown was the opposite (Fig. 3).  (e,f) tunel assays were used to detect the cell proliferation and apoptosi; (g,h) A549-DDP cells were co-transfected with pcDNA-KIF2C and miR-186-3p mimics, and A549 cells were co-transfected with si-KIF2C and miR-186-3p inhibitors. After transfection, Western blot were used to detect the changes of β-catenin, p-GSK-3β and p-AKT; (i,l,m) the effects of KIF2C on migration and invasion were evaluated by the wound healing and transwell assays (*P < 0.05, **P < 0.01, and ***P < 0.001). www.nature.com/scientificreports/ miR-186-3p targeted KIF2C regulation. The StarBase database was chosen to predict the miRNAs that could control KIF2C for a further understanding of the upstream regulatory mechanisms of KIF2C. It was demonstrated that miR-186-3p and KIF2C 3'UTR have binding sites (Fig. 4a, Supplementary file). To verify the targeting relationship between KIF2C 3'UTR and miR-186-3p, we performed a dual luciferase reporter assay. According to the results, overexpression of miR-186-3p reduced the luciferase activity in KIF2C WT cells, but had no discernible effect on KIF2C MUT cells (Fig. 4b). Compared with BEAS-2B cells, miR-186-3p expression was lower in lung cancer cells (Fig. 4c).
The results showed that A549-DDP cells transfected with miR-186-3p mimics and A549 cells transfected with si-KIF2C exhibited a higher rate of apoptosis than control cells (Fig. 5e,f). After transfection, western blot was conducted to investigate the effect of miR-186-3p/KIF2C axis on AKT-GSK3β-β-catenin pathway. We found that the levels of β-catenin, p-GSK-3β and p-AKT decreased in A549-DDP cells transfected with miR-186-3p mimics and A549 cells transfected with si-KIF2C compared with those of controls, which indicated that the AKT-GSK3ββ-catenin pathway was inhibited by miR-186-3p/KIF2C axis (Fig. 5g,h). Subsequently, we investigated the effect of miR-186-3p/KIF2C axis on cell proliferation, migration and invasion in NSCLC by performing transwell, colony formation and wound healing assays. The findings revealed that miR-186-3p inhibitors promoted proliferation, migration and invasion of A549 cell compared with the control group, whereas miR-186-3p mimics impeded above features of A549-DDP cells (Fig. 5i,l,m). Overall, these findings suggest that miR-186-3p/KIF2C axis play an important role in biologically malignant behaviors of NSCLC cells.

Discussion
Incidence and mortality from cancer are most frequently caused by lung cancer worldwide 14 . NSCLC is distinguished by slow cancer cell growth, metastasis, and development 15 . One of the most important symptoms of NSCLC is the metastasis of tumor cells 16 . Therefore, inhibiting tumor cell proliferation and metastasis has a www.nature.com/scientificreports/ huge auxiliary effect on patients with lung cancer 15 . Researchers found that KIF2C was highly expressed in liver cancer and thyroid cancer, and was a landmark tumor-promoting factor 3,17 . However, the activation mechanism of KIF2C in NSCLC has not been investigated 18 . Therefore, we designed this experiment to explore a new mechanism for restricting the proliferation and metastasis of NSCLC. The study of cancer uses three main approaches: fresh tumor tissue, animal models, and cell cultures. They all have advantages and disadvantages. However, each tumor tissue contains varying amounts of non-malignant cells, and there are restrictions on the acquisition and use of these cells. Animal experiments need to consider species differences. These reasons have made cancer cell lines an important tool for studying lung cancer. Currently an estimated 300-400 human lung cancer cell lines have been established, including small cell (SCLC) and NSCLC. These cell lines have been widely used in the scientific community all over the world and their research has generated more than a thousand citations, and the research results have played a crucial role in revealing the pathogenesis of lung cancer 19 . We found that KIF2C was closely related to chemotherapy resistance of breast cancer through the literature 20 . The latest research has found that KIF2C, as a stemness related genes, may affect the efficacy of chemotherapy and immunotherapy by regulating the tumor microenvironment in LUAD 21 . This also reflects the innovation of our research. In this paper, we presented experimental proof that, in comparison to BEAS-2B, KIF2C was significantly expressed in various kinds of lung cancer cell lines such as LUAD and LUSC. The results of cell experiments showed that KIF2C overexpression significantly enhanced cell proliferation, migration, and invasion while inhibiting cell apoptosis when compared to the control group, whereas KIF2C knockdown prevented cell proliferation, migration and invasion, and enhanced cell apoptosis. The above results illustrated the critical role of KIF2C in the proliferation and metastasis of NSCLC. Meanwhile, according to Western blotting results, overexpression of KIF2C raised the levels of β-catenin, p-GSK-3β, and p-AKT, but downregulation of the protein had the reverse effect. The results demonstrated that KIF2C may contribute to NSCLC growth and metastasis by triggering the AKT-GSK3β-β-catenin signaling pathway. Many miRNAs act as tumor suppressors or oncogenes in most cancers, and some have tissue-specific expression patterns 8,22 . Researchers found that miR-186 suppressed growth, migration, and invasion of NSCLC [23][24][25] . MiR-186-3p is involved in a variety of cancer pathogenesis functions 26 . Lu et al. found that miR-186-3p can reduce tamoxifen resistance in breast cancer cells through the EREG axis and can prevent the onset and progression of cervical cancer via targeting IGF1 27 . There are also reports that miR-186-3p can block tumor growth in lung cancer 28 .
We experimentally verified the targeting link between miR-186-3p and KIF2C 3'UTR, and the results suggested that miR-186-3p may control KIF2C expression. In addition to directly inhibiting NSCLC growth and metastasis, miR-186-3p can also reverse the NSCLC-promoting effects of KIF2C. The relationship between KIF2C and miR-186-3p has been shown in the schematic diagram (Fig. 6).
In conclusion, KIF2C is widely expressed in NSCLC, which can promote the proliferation and metastasis of NSCLC. Mir-186-3p can slow down the deterioration of NSCLC by inhibiting the activation of AKT-GSK3ββ-catenin signaling pathway by KIF2C. As a result, we believe KIF2C could be a possible target for NSCLC treatment (Supplementary Information).